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1. Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design

   https://doi.org/10.1002/pro.4543  

   Srivastava, Ayush K.; Scalcione, Lucas J.; Arosio, Paolo; Bou‐Abdallah, Fadi (December 2022, Protein Science)

   Abstract Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co‐assemble in different H and L ratios to form 24‐mer shell‐like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structure–function relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L‐rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1°C) compared to their H‐ferritin homologues (Tm = 93 ± 1°C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron‐induced protein thermal destabilization effect (ΔT_mup to 20°C) is observed. To our knowledge, this is the first report of recombinant human homo‐ and hetero‐polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L‐rich ferritins may have great potential for biotechnological applications. 

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2. Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen

   https://doi.org/10.3390/ijms23031054  

   Popovic, Grega; Kirby, Nicholas C.; Dement, Taylor C.; Peterson, Kristine M.; Daub, Caroline E.; Belcher, Heather A.; Guthold, Martin; Offenbacher, Adam R.; Hudson, Nathan E. (February 2022, International Journal of Molecular Sciences)

   Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial. 

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3. Sustained Release of Recombinant Human Growth Hormone from Bioresorbable Poly(ester urea) Nanofibers

   https://doi.org/10.1021/acsmacrolett.7b00334  

   Gao, Yaohua; Xu, Yanyi; Land, Adam; Harris, Justin; Policastro, Gina M.; Childers, Erin P.; Ritzman, Todd; Bundy, Joshua; Becker, Matthew L. (July 2017, ACS Macro Letters)
4. Advancing to Recombinant Heparin

   Thacker, Bryan; Glass, Charles; Sharfstein, Susan (July 2021, American pharmaceutical review)

   null (Ed.)

   Recombinant heparin is produced by genetically engineered cells and provides an alternative to the current animal derived heparin. 

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5. Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

   https://doi.org/10.7717/peerj.3512  

   Bajinting, Adam; Ng, Ho Leung (January 2017, PeerJ)

   Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs) as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity. 

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